The Acute Lung Injury/Acute Respiratory Distress Syndrome (ALI/ARDS) is a devastating consequence of systemic inflammatory conditions such as sepsis that afflicts almost 200,000 people a year in the US with 75,000 deaths. The hallmark of ALI is inflammation-induced disruption of the endothelial cell (EC) barrier that lines the pulmonary vasculature, resulting in leakage of fluid, protein, and cells into the airspaces of the lung. Our laboratory has extensively studied the mechanisms involved in maintaining and enhancing EC barrier function as a tool for identifying possible therapeutic targets. The current paradigm of EC barrier regulation suggests a balance exists between barrier-disrupting cellular contractile forces and barrier-protective cell-cell and cell-matrix tethering forces. Both competing forces in this model are intimately linked to the actin-based endothelial cytoskeleton by a variety of actin-binding proteins. Our work has defined an essential role for the actin-binding protein, cortactin, in the resolution phase of vascular permeability with this critical function occurring via EC cytoskeletal rearrangement. Very little is known about the mechanisms governing recovery of EC barrier function following injury. Thus, cortactin is an attractive molecular target for novel therapies and warrants the intense structure/function studies we propose in this application. With this background, the PI proposes to investigate the hypothesis that cortactin regulates EC cytoskeletal rearrangements that result in altered barrier function during ALI syndromes. In SA#1 we will mechanistically characterize the key portions of the cortactin molecule involved in barrier regulation through the use of molecular biology and proteomic techniques utilizing in vitro models of barrier disruption (e.g., thrombin, TGFpl) to focus on cortactin's role during the barrier recovery phase. Transgenic animal models of ALI will extend these studies in vivo. In SA#2 we will examine the role of cortactin in cortical actin and junctional protein rearrangements that regulate pulmonary endothelial barrier function using novel atomic force microscopy (AFM) and other techniques to functionally characterize cortactin's role in peripheral cytoskeletal rearrangements involved in barrier recovery, focusing on cortical actin structures, junctional complex formation, and lipid raft signaling. In SA#3 we will characterize the functional consequences of an ALI- associated coding single nucleotide polymorphism (SNP) we have identified in the cortactin gene through a combination of molecular biology and proteomic techniques. This aim will determine the mechanistic effects of this ALI-associated SNP on cortactin function as it pertains to endothelial permeability and barrier recovery using the in vitro and transgenic animal techniques described above.